Johannes A. M. te Poele

Single-Dose and Fractionated Irradiation Promote Initiation and Progression of Atherosclerosis and Induce an Inflammatory Plaque Phenotype in ApoE−/− Mice

PURPOSE Increased risk of atherosclerosis and stroke has been demonstrated in patients receiving radiotherapy for Hodgkins lymphoma and head-and-neck cancer. We previously showed that 14 Gy to the carotid arteries of hypercholesterolemic ApoE(-/-) mice resulted in accelerated development of macrophage-rich, inflammatory atherosclerotic lesions. Here we investigate whether clinically relevant fractionated irradiation schedules and lower single doses also predispose to an inflammatory plaque phenotype. METHODS AND MATERIALS ApoE(-/-) mice were given 8 or 14 Gy, or 20 x 2.0 Gy in 4 weeks to the neck, and the carotid arteries were subsequently examined for presence of atherosclerotic lesions, plaque size, and phenotype. RESULTS At 4 weeks, early atherosclerotic lesions were found in 44% of the mice after single doses of 14 Gy but not in age-matched controls. At 22 to 30 weeks after irradiation there was a twofold increase in the mean number of carotid lesions (8-14 Gy and 20 x 2.0 Gy) and total plaque burden (single doses only), compared with age-matched controls. The majority of lesions seen at 30 to 34 weeks after fractionated irradiation or 14-Gy single doses were granulocyte rich (100% and 63%, respectively), with thrombotic features (90% and 88%), whereas these phenotypes were much less common in age-matched controls or after a single dose of 8 Gy. CONCLUSIONS We showed that fractionated irradiation accelerated the development of atherosclerosis in ApoE(-/-) mice and predisposed to the formation of an inflammatory, thrombotic plaque phenotype.

Irradiation induced modest changes in murine cardiac function despite progressive structural damage to the myocardium and microvasculature

BACKGROUND Radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiotoxicity, but the underlying mechanisms are unclear. METHODS Single doses of 2, 8, or 16 Gy were delivered to the hearts of mice and damage was evaluated at 20, 40, and 60 weeks, relative to age matched controls. Single photon emission computed tomography (SPECT/CT) and ultrasound were used to measure cardiac geometry and function, which was related to histo-morphology and microvascular damage. RESULTS Gated SPECT/CT and ultrasound demonstrated decreases in end diastolic and systolic volumes, while the ejection fraction was increased at 20 and 40 weeks after 2, 8, and 16 Gy. Cardiac blood volume was decreased at 20 and 60 weeks after irradiation. Histological examination revealed inflammatory changes at 20 and 40 weeks after 8 and 16 Gy. Microvascular density in the left ventricle was decreased at 40 and 60 weeks after 8 and 16 Gy, with functional damage to remaining microvasculature manifest as decreased alkaline phosphatase (2, 8, and 16 Gy), increased von Willebrand Factor and albumin leakage from vessels (8 and 16 Gy), and amyloidosis (16 Gy). 16 Gy lead to sudden death between 30 and 40 weeks in 38% of mice. CONCLUSIONS Irradiation with 2 and 8 Gy induced modest changes in murine cardiac function within 20 weeks but this did not deteriorate further, despite progressive structural and microvascular damage. This indicates that heart function can compensate for significant structural damage, although higher doses, eventually lead to sudden death.

Identification of Differentially Expressed Genes in Mouse Kidney after Irradiation using Microarray Analysis

Abstract Kruse, J. J. C. M., te Poele, J. A. M., Velds, A., Kerkhoven, R. M., Boersma, L. J., Russell, N. S. and Stewart, F. A. Identification of Differentially Expressed Genes in Mouse Kidney after Irradiation using Microarray Analysis. Radiat. Res. 161, 28–38 (2004). Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. The molecular mechanisms underlying the development of radiation-induced nephropathy are unclear. Given the complexity of radiation-induced responses, microarrays may offer new opportunities to identify a wider range of genes involved in the development of radiation injury. The aim of the present study was to determine whether microarrays are a useful tool for identifying time-related changes in gene expression and potential mechanisms of radiation-induced nephropathy. Microarray experiments were performed using amplified RNA from irradiated mouse kidneys (1 × 16 Gy) and from sham-irradiated control tissue at different intervals (1–30 weeks) after irradiation. After normalization procedures (using information from straight-color, color-reverse and self-self experiments), the differentially expressed genes were identified. Control and repeat experiments were done to confirm that the observations were not artifacts of the array procedure (RNA amplification, probe synthesis, hybridizations and data analysis). To provide independent confirmation of microarray data, semi-quantitative PCR was performed on a selection of genes. At 1 week after irradiation (before the onset of vascular and functional damage), 16 genes were significantly up-regulated and 9 genes were down-regulated. During the period of developing nephropathy (10 to 20 weeks), 31 and 42 genes were up-regulated and 9 and 4 genes were down-regulated. At the later time of 30 weeks, the vast majority of differentially expressed genes (191 out of 203) were down-regulated. Potential genes of interest included TSA-1 (also known as Ly6e) and Jagged 1 (Jag1). Increased expression of TSA-1, a member of the Ly-6 family, has previously been reported in response to proteinuria. Jagged 1, a ligand for the Notch receptor, is known to play a role in angiogenesis, and is particularly interesting in the context of radiation-induced vascular injury. The present study demonstrates the potential of microarrays to identify changing patterns of gene expression in irradiated kidney. Further studies will be required to evaluate functional involvement of these genes in vascular-mediated normal tissue injury.

Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys

Abstract Kruse, J. J. C. M., Floot, B. G. J., Te Poele, J. A. M., Russell, N. S. and Stewart, F. A. Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys. Radiat. Res. 171, 188–197 (2009). The purpose of this study was to investigate the long-term effects of radiation-induced alterations in TGF-β signaling pathways with respect to the development of vascular damage in the irradiated kidney. Total RNA was isolated from mouse kidneys at 1–30 weeks after irradiation, and quantitative real-time PCR analyses were performed for TGF-β receptors (ALK1, ALK5, endoglin), downstream mediators (Smad7, CTGF), and downstream targets (PAI-1 and Id-1). Expression of endoglin and Smad7 protein as well as nucleo-cytoplasmic distribution of phospho Smad 2/3 and phospho Smad 1/5 was analyzed by immunohistochemistry. Radiation caused a rapid and persistent increase in expression of TGF-β receptors and mediators from 1–30 weeks after treatment. Expression of Id-1, a downstream target of endothelial cell specific receptor ALK1, was transiently increased (1–10 weeks after irradiation) but returned to control levels at later times. Expression of PAI-1, a downstream target of ALK5, increased progressively from 10–30 weeks after irradiation. These results show that radiation activated TGF-β signaling pathways in the kidney and shifted the balance in favor of ALK5 signaling, which generally inhibits endothelial cell proliferation and migration. We hypothesize that prolonged activation of ALK5 signaling and relative suppression of ALK1 signaling may provide an explanation for the telangiectatic phenotype observed in irradiated kidneys.

Irradiation induces different inflammatory and thrombotic responses in carotid arteries of wildtype C57BL/6J and atherosclerosis-prone ApoE / mice

BACKGROUND AND PURPOSE We have previously shown that irradiation to the carotid arteries of hypercholesterolemic ApoE(-/-) mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions. We now investigated the mechanism underlying the development of radiation-induced atherosclerosis. MATERIALS AND METHODS ApoE(-/-) and wildtype C57BL/6J mice received 0, 8 or 14 Gy to the neck and the carotid arteries were harvested 1 day, 1 or 4 weeks later. Immunohistochemical stainings were performed to evaluate well-known inflammatory and thrombotic molecules. A hypothesis-generating approach was used to compare gene expression profiles of irradiated and unirradiated carotid arteries. RESULTS Basal levels of endothelial VCAM-1 and thrombomodulin immunoexpression were higher in ApoE(-/-) mice than in C57BL/6J mice. At 1 week after 14 Gy VCAM-1 immunoexpression was decreased in ApoE(-/-) mice, whereas ICAM-1 immunoexpression was decreased at 1 and 4 weeks after 14 Gy in C57BL/6J mice. Thrombomodulin and tissue factor immunoexpression were elevated at 4 weeks after 14 Gy in ApoE(-/-) mice and reduced in C57BL/6J mice. There were no changes in immunoexpression of eNOS, MCP-1 or endoglin. Several canonical pathways were differentially expressed after irradiation, including tight junction pathways, leukocyte extravasation signaling and PI3K/AKT signaling. CONCLUSION ApoE(-/-) and C57BL/6J mice respond differently to irradiation. The thrombotic pathways were activated after irradiation in ApoE(-/-) mice only. Genes involved in tight junction regulation were up-regulated in ApoE(-/-) mice and decreased in C57BL/6J mice. These factors may have contributed to fatty-streak formation in ApoE(-/-) mice.

Radiation nephropathy--the link between functional damage and vascular mediated inflammatory and thrombotic changes.

The extent of radiation-induced nephropathy, which develops progressively over periods of months to years after treatment, is strongly influenced by both total dose and dose per fraction. In this study we examined the relationship between the early expression of various thrombotic and inflammatory markers of endothelial cell (EC) damage in irradiated mouse kidneys and the subsequent development of nephropathy. Decreased levels of glomerular ADPase and increased levels of glomerular Vwf were seen from 4 or 20 weeks after irradiation, respectively. These pro-thrombotic changes were associated with increased fibrin/fibrinogen deposits, indicative of microthrombus formation, at later times. These events were, however, not sensitive to changes in total dose or dose per fraction, therefore they cannot be quantitatively linked to the development of radiation nephropathy. Increased leucocyte invasion of the renal cortex was also seen after irradiation; this was quantitatively dependent on both total dose and dose per fraction. Linear quadratic analysis of the leucocyte dose-response curves yielded an alpha/beta ratio of 7.7 Gy, which is significantly greater than the alpha/beta ratio or 2.7 Gy determined for nephropathy, indicating less fractionation sensitivity for the inflammatory response. We conclude that inflammatory changes contribute to, but do not entirely explain, radiation nephropathy. The role of thrombotic changes is less clear.The extent of radiation-induced nephropathy, which develops progressively over periods of months to years after treatment, is strongly influenced by both total dose and dose per fraction. In this study we examined the relationship between the early expression of various thrombotic and inflammatory markers of endothelial cell (EC) damage in irradiated mouse kidneys and the subsequent development of nephropathy. Decreased levels of glomerular ADPase and increased levels of glomerular Vwf were seen from 4 or 20 weeks after irradiation, respectively. These pro-thrombotic changes were associated with increased fibrin fibrinogen deposits, indicative of microthrombus formation, at later times. These events were, however, not sensitive to changes in total dose or dose per fraction, therefore they cannot be quantitatively linked to the development of radiation nephropathy. Increased leucocyte invasion of the renal cortex was also seen after irradiation; this was quantitatively dependent on both total dose and dose per fraction. Linear quadratic analysis of the leucocyte dose-response curves yielded an f / g ratio of 7.7 Gy, which is significantly greater than the f / g ratio or 2.7 Gy determined for nephropathy, indicating less fractionation sensitivity for the inflammatory response. We conclude that inflammatory changes contribute to, but do not entirely explain, radiation nephropathy. The role of thrombotic changes is less clear.

Anti-inflammatory and anti-thrombotic intervention strategies using atorvastatin, clopidogrel and knock-down of CD40L do not modify radiation-induced atherosclerosis in ApoE null mice

BACKGROUND AND PURPOSE We previously showed that irradiating the carotid arteries of ApoE(-/-) mice accelerated the development of macrophage-rich, inflammatory and thrombotic atherosclerotic lesions. In this study we investigated the potential of anti-inflammatory (atorvastatin, CD40L knockout) and anti-thrombotic (clopidogrel) intervention strategies to inhibit radiation-induced atherosclerosis. MATERIAL AND METHODS ApoE(-/-) mice were given 0 or 14 Gy to the neck and the carotid arteries were harvested at 4 or 28 weeks after irradiation. Atorvastatin (15 mg/kg/day) or clopidogrel (20 mg/kg/day) was given in the chow; control groups received regular chow. Clopidogrel inhibited platelet aggregation by 50%. CD40L(-/-)/ApoE(-/-) and ApoE(-/-) littermates were also given 0 or 14 Gy to the neck and the carotid arteries were harvested after 30 weeks. RESULTS Clopidogrel decreased MCP-1 expression in the carotid artery at 4 weeks after irradiation. Expression of VCAM-1, ICAM-1, thrombomodulin, tissue factor and eNOS was unchanged in atorvastatin and clopidogrel-treated mice. Neither drug inhibited either age-related or radiation-induced atherosclerosis. Furthermore, loss of the inflammatory mediator CD40L did not influence the development of age-related and radiation-induced atherosclerosis. CONCLUSIONS The effects of radiation-induced atherosclerosis could not be circumvented by these specific anti-inflammatory and anti-coagulant therapies. This suggests that more effective drug combinations may be required to overcome the radiation stimulus, or that other underlying mechanistic pathways are involved compared to age-related atherosclerosis.

NO-Donating Aspirin and Aspirin Partially Inhibit Age-Related Atherosclerosis but Not Radiation-Induced Atherosclerosis in ApoE Null Mice

Background We previously showed that irradiation to the carotid arteries of ApoE−/− mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. In this study we investigated the potential of anti-inflammatory and anti-coagulant intervention strategies to inhibit age-related and radiation-induced atherosclerosis. Methodology/Principal Findings ApoE−/− mice were given 0 or 14 Gy to the neck and the carotid arteries and aortic arches were harvested at 4 or 30 weeks after irradiation. Nitric oxide releasing aspirin (NCX 4016, 60 mg/kg/day) or aspirin (ASA, 30 or 300 mg/kg/day) were given continuously in the chow. High dose ASA effectively blocked platelet aggregation, while the low dose ASA or NCX 4016 had no significant effect on platelet aggregation. High dose ASA, but not NCX 4016, inhibited endothelial cell expression of VCAM-1 and thrombomodulin in the carotid arteries at 4 weeks after irradiation; eNOS and ICAM-1 levels were unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total number of lesions and the number of initial macrophage-rich lesions in the carotid arteries of unirradiated mice, but these effects were not seen in the brachiocephalic artery of the aortic arch (BCA). In contrast, high dose ASA lead to a decrease in the number of initial lesions in the BCA, but not in the carotid artery. Both high dose ASA and NCX 4016 reduced the collagen content of advanced lesions and increased the total plaque burden in the BCA of unirradiated mice. At 30 weeks after irradiation, neither NCX 4016 nor ASA significantly influenced the number or distribution of lesions, but high dose ASA lead to formation of collagen-rich “stable” advanced lesions in carotid arteries. The total plaque area of the irradiated BCA was increased after ASA, but the plaque burden was very low compared with the carotid artery. Conclusions/Significance The development and characteristics of radiation-induced atherosclerosis varied between different arteries but could not be circumvented by anti-inflammatory and anti-coagulant therapies. This implicates other underlying mechanistic pathways compared to age-related atherosclerosis.

Irradiation of existing atherosclerotic lesions increased inflammation by favoring pro-inflammatory macrophages.

BACKGROUND AND PURPOSE Recent studies have shown an increased incidence of localized atherosclerosis and subsequent cardiovascular events in cancer patients treated with thoracic radiotherapy. We previously demonstrated that irradiation accelerated the development of atherosclerosis and predisposed to an inflammatory plaque phenotype in young hypercholesterolemic ApoE(-/-) mice. However, as older cancer patients already have early or advanced stages of atherosclerosis at the time of radiotherapy, we investigated the effects of irradiation on the progression of existing atherosclerotic lesions in vivo. MATERIAL AND METHODS ApoE(-/-) mice (28 weeks old) received local irradiation with 14 or 0 Gy (sham-treated) at the aortic arch and were examined after 4 and 12 weeks for atherosclerotic lesions, plaque size and phenotype. Moreover, we investigated the impact of irradiation on macrophage phenotype (pro- or anti-inflammatory) and function (efferocytotic capacity, i.e. clearance of apoptotic cells) in vitro. RESULTS Irradiation of existing lesions in the aortic arch resulted in smaller, macrophage-rich plaques with intraplaque hemorrhage and increased apoptosis. In keeping with the latter, in vitro studies revealed augmented polarization toward pro-inflammatory macrophages after irradiation and reduced efferocytosis by anti-inflammatory macrophages. In addition, considerably more lesions in irradiated mice were enriched in pro-inflammatory macrophages. CONCLUSIONS Irradiation of existing atherosclerotic lesions led to smaller but more inflamed plaques, with increased numbers of apoptotic cells, most likely due to a shift toward pro-inflammatory macrophages in the plaque.

Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel

PURPOSE To assess the effects of kidney irradiation on glomerular adenosine diphosphatase (ADPase) activity and intraglomerular microthrombus formation, and their correlation to the development of renal functional impairment. METHODS AND MATERIALS C3H/HenAf-nu(+) mice were given single-dose or fractionated kidney irradiations. Glomerular ADPase activity was measured using a cerium-based histochemical method. Microthrombus formation within the glomeruli was assessed by a semiquantitative immunohistochemical analysis of fibrinogen/fibrin deposits. Renal function was assessed by the [(51)Cr]EDTA retention assay. RESULTS The ADPase activity was significantly reduced, to approximately 50% of pretreatment value, 4--40 weeks after 10--16 Gy single-dose irradiation and at 44 weeks after 20 x 2 Gy. No dose--effect relationship was found. An approximately fourfold increase in glomerular fibrinogen/fibrin staining was observed at 1 year after irradiation. This increase was not influenced by treating the mice with daily, oral clopidogrel, a platelet ADP receptor antagonist, which reduced platelet aggregation by more than 75%. Radiation-induced impairment of glomerular filtration was also not affected by the clopidogrel treatment. CONCLUSION These data indicate that irradiation significantly reduced glomerular ADPase activity, which correlated with an increased glomerular fibrinogen/fibrin deposition. We were not able to reduce these prothrombotic changes, nor to protect against radiation nephropathy, by pharmacological intervention with an ADP-receptor antagonist.